Immune Response of BALB/c Mice to Phthalate: Characterization of a New and Useful Model for Studying Immune Regulation

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Abstract
The humoral immune response to a unique haptenic determinant has been characterized at the single cell level. We report here that the hapten, phthalate, when conjugated to keyhole limpet hemocyanin (4-azophthalate-KLH) is highly immunogenic in adult BALB/c mice, and that the immunogenicity of the hapten is largely restricted to the two charged carboxylate groups on phthalate. Based upon plaque-forming cell (PFC) inhibition with increasing concentrations of free phthalate, relative affinity distribution profiles of antibody-forming cells have been established for individual mice. The antibody-forming cells in the spleens of these mice consist of five or six individual relative affinity sets. PFC inhibition studies with hybridomas secreting phthalate specific monoclonal antibodies reveal, as expected, a more restricted inhibition profile. The majority of PFC produced by the hybridomas could be assigned to one of the relative affinity sets. PFC inhibition studies employing a panel of cross-reactive phthalate analogs as inhibitors, have made it possible to segregate the splenic antibody-forming cell population further into distinct fine specificity sets. The recognized immunodominance of the two negatively charged carboxylate groups of phthalate and the rigid positioning of substituted charged groups on the phthalate analogs are unique to this hapten system and they are fundamental to the meaningful interpretation of the fine specificity data generated by the inhibition studies reported here. The ability to compartmentalize the phthalate response into relative affinity and fine specificity sets at the single cell level has made it possible for us to restrict our studies of immune regulation to a defined and manageable portion of a conventional thymic dependent B cell repertoire.